In all organisms, ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to 2′-deoxynucleotides, providing the precursors used in DNA biosynthesis and repair.1-3 The mechanism of nucleotide reduction is conserved in all RNRs and requires formation of a transient active site thiyl radical (C439., E. coli RNR numbering used throughout the text).4,5 However, the mechanism of active site thiyl radical generation, the radical initiation event, is not conserved and provides the basis for distinction between four classes of RNRs.6-9 A major unresolved mechanistic issue is that of thiyl radical formation in class I RNRs, and presumably in the recently identified class IV RNRs.
The E. coli class I RNR consists of two homodimeric subunits, α2 and β2, which form an active 1:1 complex during turnover.10-12 α2 is the business end of the complex. It contains the active site where thiyl radical-mediated nucleotide reduction occurs, as well as multiple allosteric effector binding sites which modulate substrate specificity and turnover rate.13 β2 houses the stable diferric tyrosyl radical (Y122.)14-16 cofactor that is required for formation of the transient C439. in the active site of α2.4-6 The structures of α26,17 and β218,19 have been solved and a structure containing both subunits has also been reported.20 A structure of the active α2β2 complex, however, has remained elusive. From the individual structures of α2 and β2, Uhlin and Eklund have generated a docking model of the α2β2 complex based on shape and charge complementarity and conserved residues.6 This model suggests that the Y122. in β2 is located >35 Å away from C439 in α2 (FIG. 1).21-23 Radical propagation over this long distance requires the involvement of transient amino acid intermediates.24-26 The residues proposed to participate in this pathway are universally conserved in all class I RNRs.
Evidence in support of the long distance between Y122. and C439 has recently been obtained from pulsed electron-electron double resonance spectroscopic measurements27 with a mechanism based inhibitor.28-32 The distance obtained from this study is consistent with the docking model and establishes that a large conformational change, that positions Y122. in β2 adjacent to C439 in α2, does not occur.32 
To examine the validity of the proposed pathway, site-directed mutagenesis33,34 and complementation studies35 have been carried out. These studies demonstrate that each residue in FIG. 1 plays an important role in RNR function. However, the absence of activity in these mutants precludes mechanistic investigations.33,34 At present, evidence, e.g., detailed elsewhere herein, for the involvement of only one of the proposed pathway residues, Y356, is substantial. In contrast, the roles of α2 residues Y730 and Y731 in radical propagation are still ill-defined. Mutagenesis studies have demonstrated their importance in RNR function.34,44,45 However, as with residue Y356 in β2, the inactivity of these mutants (Y730F—α2 and Y731F-α2) precluded mechanistic interrogation of the role of Y730 and Y731 in radical propagation.
What is needed in the art are methods and compositions for the site-specific replacement of an amino acid residue that is proposed to participate in radical propagation in a reductase enzyme with unnatural amino acid residue that produces a mechanistically informative mutant, e.g., a mutant that can be used to interrogate the replaced amino acid's function in radical propagation in, e.g., a reductase enzyme. The present invention provides new tools and methods for elucidating RNR reaction mechanisms.